Compounds and methods for inhibiting mrp1

ABSTRACT

The present invention relates to a compound of formula (I) which are useful for inhibiting resistant neoplasms where the resistance is conferred in part or in total by MRP1.

[0001] Along with surgery and radiotherapy, chemotherapy continues to bean effective therapy for many cancers. In fact, several types of cancer,such as Hodgkin's disease, large cell lymphoma, acute lymphocyticleukemia, testicular cancer and early stage breast cancer, are nowconsidered to be curable by chemotherapy. Other cancers such as ovariancancer, small cell lung and advanced breast cancer, while not yetcurable, are exhibiting positive response to combination chemotherapy.

[0002] One of the most important unsolved problems in cancer treatmentis drug resistance. After selection for resistance to a single cytotoxicdrug, cells may become cross resistant to a whole range of drugs withdifferent structures and cellular targets, e.g., alkylating agents,antimetabolites, hormones, platinum-containing drugs, and naturalproducts. This phenomenon is known as multidrug resistance (MDR). Insome types of cells, this resistance is inherent, while in others, suchas small cell lung cancer, it is usually acquired.

[0003] Such resistance is known to be multifactorial and is conferred byat least two proteins: the 170 kDa P-glycoprotein (MDR1) and the morerecently identified 190 kDa multidrug resistance protein (RP1). Althoughboth MDR1 and MRP1 belong to the ATP-binding cassette superfamily oftransport proteins, they are structurally very different molecules andshare less than 15% amino acid homology. Despite the structuraldivergence between the two proteins, by 1994 there were no knownconsistent differences in the resistance patterns of MDR1 and MRP1 celllines. However, the association, or lack thereof, of MRP1 and resistanceto particular oncolytics is known. See Cole, et. al., “PharmacologicalCharacterization of Multidrug Resistant MRP-transfected Human TumorCells”, Cancer Research, 54:5902-5910, 1994. Doxorubicin, daunorubicin,epirubicin, vincristine, paclitaxel, mitoxantrone, melphalan, andetoposide are substrates of MRP1, i.e., MRP1 can bind to theseoncolytics and redistribute them away from their site of action, thenucleus, and out of the cell. Id. and Marquardt, D., and Center, M. S.,Cancer Research, 52:3157, 1992.

[0004] Doxorubicin, daunorubicin, and epirubicin are members of theanthracycline class of oncolytics. They are isolates of various strainsof Streptomyces and act by inhibiting nucleic acid synthesis. Theseagents are useful in treating neoplasms of the bone, ovaries, bladder,thyroid, and especially the breast. They are also useful in thetreatment of acute lymphoblastic and myeloblastic leukemia, Wilm'stumor, neuroblastoma, soft tissue sarcoma, Hodgkin's and non-Hodgkin'slymphomas, and bronchogenic carcinoma.

[0005] Vincristine, a member of the vinca alkaloid class of oncolytics,is an isolate of a common flowering herb, the periwinkle plant (Vincarosea Linn). The mechanism of action of vincristine is still underinvestigation but has been related to the inhibition of microtubuleformation in the mitotic spindle. Vincristine is useful in the treatmentof acute leukemia, Hodgkin's disease, non-Hodgkin's malignant lymphomas,rhabdomyosarcoma, neuroblastoma, and Wilm's tumor.

[0006] Etoposide, a member of the epipodophyllotoxin class ofoncolytics, is a semisynthetic derivative of podophyllotoxin. Etoposideacts as a topoisomerase inhibitor and is useful in the therapy ofneoplasms of the testis, and lung.

[0007] It is presently unknown what determines whether a cell line willacquire resistance via a MDR1 or MRP1 mechanism. Due to the tissuespecificity of these transporters and/or in the case where one mechanismpredominates or is exclusive, it would be useful to have a selectiveinhibitor of that one over the other. Furthermore, when administering adrug or drugs that are substrates of either protein, it would beparticularly advantageous to coadminister an agent that is a selectiveinhibitor of that protein. It is, therefore, desirable to providecompounds that are selective inhibitors of MDR1 or MRP1.

[0008] The present invention relates to a compound of formula I:

[0009] wherein:

[0010] R¹ is hydrogen, optionally substituted C₁-C₄ alkyl,(CH₂)_(n)C(O)R², (C₁-C₄ alkyl)NH₂, (CH₂)_(n)NHC(O)R³, (optionallysubstituted C₁-C₄ alkyl)-optionally substituted phenyl, or optionallysubstituted heterocycle;

[0011] n is 0, 1, or 2;

[0012] p is 0, 1, 2, 3, or 4;

[0013] R² is C₁-C₄ alkoxy, (optionally substituted C₁-C₄alkyl)-optionally substituted phenyl, (CH₂)_(p)-optionally substitutedheterocycle, NHR⁴, or (CH₂)_(p)-O-optionally substituted heterocycle;

[0014] R³ is C₁-C₄ alkoxy, optionally substituted phenyl, (optionallysubstituted C₁-C₄ alkyl)-optionally substituted phenyl, or(CH₂)_(p)-optionally substituted heterocycle;

[0015] R⁴ is (CH₂)_(p)-optionally substituted phenyl or(CH₂)_(p)-optionally substituted heterocycle; or a pharmaceutical saltthereof.

[0016] The present invention further relates to a method of inhibitingMRP1 in a mammal which comprises administering to a mammal in needthereof an effective amount of a compound of formula I, or apharmaceutical salt thereof.

[0017] In another embodiment, the present invention relates to a methodof inhibiting a resistant neoplasm, or a neoplasm susceptible toresistance in a mammal which comprises administering to a mammal in needthereof an effective amount of a compound of formula I, or apharmaceutical salt thereof, in combination with an effective amount ofan oncolytic agent.

[0018] Furthermore, this invention provides the use of a compound ofFormula I for the manufacture of a medicament for the inhibition ofMRP1. This invention also provides the use of a compound of Formula Ifor the manufacture of a medicament for the inhibition of a resistantneoplasm.

[0019] The present invention also relates to a pharmaceuticalformulation comprising a compound of formula I, or a pharmaceutical saltthereof, in combination with one or more oncolytics, pharmaceuticalcarriers, diluents, or excipients therefor.

[0020] The current invention concerns the discovery that a select groupof compounds, those of formula I, are selective inhibitors of multidrugresistant protein (MRP1) and are thus useful in treating MRP1 conferredmultidrug resistance (MDR) in a resistant neoplasm and a neoplasmsusceptible to resistance.

[0021] The terms “inhibit” as it relates to MRP1 and “inhibiting MRP1”refer to prohibiting, alleviating, ameliorating, halting, restraining,slowing or reversing the progression of, or reducing MRP1's ability toredistribute an oncolytic away from the oncolytic's site of action, mostoften the neoplasm's nucleus, and out of the cell.

[0022] As used herein, the term “effective amount of a compound offormula I” refers to an amount of a compound of the present inventionwhich is capable of inhibiting MRP1. The term “effective amount of anoncolytic” refers to an amount of oncolytic capable of inhibiting aneoplasm, resistant or otherwise.

[0023] The term “inhibiting a resistant neoplasm, or a neoplasmsusceptible to resistance” refers to prohibiting, halting, restraining,slowing or reversing the progression of, reducing the growth of, orkilling resistant neoplasms and/or neoplasms susceptible to resistance.

[0024] The term “resistant neoplasm” refers to a neoplasm that isresistant to chemotherapy where that resistance is conferred in part, orin total, by MRP1. Such neoplasms include, but are not limited to,neoplasms of the bladder, bone, breast, lung(small-cell), testis, andthyroid and also includes more particular types of cancer such as, butnot limited to, acute lymphoblastic and myeloblastic leukemia, Wilm'stumor, neuroblastoma, soft tissue sarcoma, Hodgkin's and non-Hodgkin'slymphomas, and bronchogenic carcinoma.

[0025] A neoplasm that is “susceptible to resistance” is a neoplasmwhere resistance is neither inherent nor currently present but can beconferred by MRP1 after chemotherapy begins. Thus, the methods of thisinvention encompass a prophylactic and therapeutic administration of acompound of formula I.

[0026] The term “chemotherapy” refers to the use of one or moreoncolytics where at least one oncolytic is a substrate of MRP1. A“substrate of MRP1” is an oncolytic that binds to MRP1 and isredistributed away from the oncolytics site of action, (the neoplasm'snucleus) and out of the cell, thus, rendering the therapy lesseffective.

[0027] The terms “treat” or “treating” bear their usual meaning whichincludes preventing, prohibiting, alleviating, ameliorating, halting,restraining, slowing or reversing the progression, or reducing theseverity of MRP1 derived drug resistance in a multidrug resistant tumor.

[0028] In the general formulae of the present document, the generalchemical terms have their usual meanings. For example, the term “C₁-C₄alkyl” refers to methyl, ethyl, propyl, isopropyl, cyclopropyl, butyl,cyclobutyl, s-butyl, and t-butyl. The term “C₁-C₆ alkyl” refers to amonovalent, straight, branched, or cyclic saturated hydrocarboncontaining from 1 to 6 carbon atoms and includes C₁-C₄ alkyl groups. Inaddition, C₁-C₆ alkyl also includes, but is not limited to, cyclopentyl,pentyl, hexyl, cyclohexyl, and the like.

[0029] The term “optionally substituted C₁-C₄ alkyl” refers to a C₁-C₄alkyl optionally substituted 1 time with a hydroxy group.

[0030] The terms “C₁-C₄ alkoxy” and “C₁-C₆ alkoxy” refer to moieties ofthe formula O—(C₁-C₄ alkyl) and O—(C₁-C₆ alkyl) respectively.

[0031] The term “halo” or “halide” refers to fluoro, chloro, bromo, andiodo.

[0032] The term “optionally substituted phenyl” refers to a phenyl ringoptionally substituted 1 or 3 times independently with a C₁-C₆ alkyl,C₁-C₄ alkoxy, halo, benzyl, phenyl, trifluoromethyl, or an oxo group.

[0033] The term “heterocycle” refers to a monovalent, saturated,unsaturated, or aromatic mono cyclic or fused ring system of 5 to 7total atoms respectively containing 1 to 3 heteroatoms selectedindependently from oxygen, sulfur, and nitrogen.

[0034] The term “optionally substituted heterocycle” refers to aheterocycle ring optionally substituted 1 or 2 times independently witha C₁-C₆ alkyl, C₁-C₄ alkoxy, halo, benzyl, phenyl, trifluoromethyl, oran oxo group.

[0035] The term “protecting group” (Pg) refers to an amino protectinggroup or a hydroxy protecting group. The species of protecting groupwill be evident from whether the “Pg” group is attached to a nitrogenatom (amino protecting group) or attached to an oxygen atom (hydroxyprotecting group).

[0036] The term “amino protecting group” as used in this specificationrefers to a substituent(s) of the amino group commonly employed to blockor protect the amino functionality while reacting other functionalgroups on the compound. Examples of such amino-protecting groups includethe formyl group, the trityl group, the phthalimido group, the acetylgroup, the trichloroacetyl group, the chloroacetyl, bromoacetyl, andiodoacetyl groups, urethane-type blocking groups such asbenzyloxycarbonyl, 9-fluorenylmethoxycarbonyl (“FMOC”), and the like;and like amino protecting groups. The species of amino protecting groupemployed is not critical so long as the derivatized amino group isstable to the condition of subsequent reaction(s) on other positions ofthe molecule and can be removed at the appropriate point withoutdisrupting the remainder of the molecule. Similar amino protectinggroups used in the cephalosporin, penicillin, and peptide arts are alsoembraced by the above terms. Further examples of groups referred to bythe above terms are described by T. W. Greene, “Protective Groups inOrganic Synthesis”, John Wiley and Sons, New York, N.Y., 1991, Chapter7. This book shall hereafter be referred to as “Greene”. A preferredamino protecting group is t-butyloxycarbonyl.

[0037] The term “hydroxy protecting group” denotes a group understood byone skilled in the organic chemical arts of the type described inChapter 2 of Greene. Representative hydroxy protecting groups include,for example, ether groups including methyl and substituted methyl ethergroups such as methyl ether, methoxymethyl ether, methylthiomethylether, tert-buylthiomethyl ether, (phenyldimethylsilyl)methoxy-methylether, benzyloxymethyl ether, p-methoxybenzyloxy-methyl ether, andtert-butoxymethyl ether; substituted ethyl ether groups such asethoxyethyl ether, 1-(2-chloroethoxy)-ethyl ether,2,2,2-trichloroethoxymethyl ether, and 2-(trimethylsilyl)ethyl ether;isopropyl ether groups; phenyl and substituted phenyl ether groups suchas phenyl ether, p-chlorophenyl ether, p-methoxyphenyl ether, and2,4dinitrophenyl ether; benzyl and substituted benzyl ether groups suchas benzyl ether, p-methoxybenzyl ether, o-nitrobenzyl ether, and2,6-dichlorobenzyl ether; and alkylsilyl ether groups such as trimethyl-triethyl- and triisopropylsilyl ethers, mixed alkylsilyl ether groupssuch as dimethylisopropylsilyl ether, and diethylisopropylsilyl ether;and ester protecting groups such as formate ester, benzylformate ester,mono-, di-, and trichloroacetate esters, phenoxyacetate ester, andp-chlorophenoxyacetate and the like. The species of hydroxy protectinggroup employed is not critical so long as the derivatized hydroxy groupis stable to the conditions of subsequent reaction(s) on other positionsof the intermediate molecule and can be selectively removed at theappropriate point without disrupting the remainder of the moleculeincluding any other hydroxy protecting group(s).

[0038] In general, the term “pharmaceutical” when used as an adjectivemeans substantially non-toxic to living organisms. For example, the term“pharmaceutical salt” as used herein, refers to salts of the compoundsof formula I which are substantially non-toxic to living organisms. See,e.g., Berge, S. M, Bighley, L. D., and Monkhouse, D. C., “PharmaceuticalSalts”, J. Pharm. Sci., 66:1, 1977. Typical pharmaceutical salts includethose salts prepared by reaction of the compounds of formula I with aninorganic or organic acid or base. Such salts are known as acid additionor base addition salts respectively. These pharmaceutical saltsfrequently have enhanced solubility characteristics compared to thecompound from which they are derived, and thus are often more amenableto formulation as liquids or emulsions.

[0039] The term “acid addition salt” refers to a salt of a compound offormula I prepared by reaction of a compound of formula I with a mineralor organic acid. For exemplification of pharmaceutical acid additionsalts see, e.g., Berge, S. M, Bighley, L. D., and Monkhouse, D. C., J.Pharm. Sci., 66:1, 1977. Since compounds of this invention can be basicin nature, they accordingly react with any of a number of inorganic andorganic acids to form pharmaceutical acid addition salts.

[0040] The pharmaceutical acid addition salts of the invention aretypically formed by reacting the compound of formula I with an equimolaror excess amount of acid. The reactants are generally combined in amutual solvent such as diethylether, tetrahydrofuran, methanol, ethanol,isopropanol, benzene, and the like. The salts normally precipitate outof solution within about one hour to about ten days and can be isolatedby filtration or other conventional methods.

[0041] Acids commonly employed to form acid addition salts are inorganicacids such as hydrochloric acid, hydrobromic acid, hydroiodic acid,sulfuric acid, phosphoric acid, and the like, and organic acids, such asp-toluenesulfonic acid, methanesulfonic acid, oxalic acid,p-bromophenylsulfonic acid, carbonic acid, succinic acid, citric acid,benzoic acid, acetic acid and the like. Examples of suchpharmaceutically acceptable salts thus are the sulfate, pyrosulfate,bisulfate, sulfite, bisulfite, phosphate, monohydrogenphosphate,dihydrogenphosphate, metaphosphate, pyrophosphate, chloride, bromide,iodide, acetate, propionate, decanoate, caprylate, acrylate, formate,isobutyrate, caproate, heptanoate, propiolate, oxalate, malonate,succinate, suberate, sebacate, fumarate, maleate, butyne-1,4-dioate,hexyne-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate,dinitrobenzoate, hydroxybenzoate, methoxybenzoate, phthalate, sulfonate,xylenesulfonate, phenylacetate, phenylpropionate, phenylbutyrate,citrate, lactate, β-hydroxybutyrate, glycollate, tartrate,methanesulfonate, propanesulfonate, 1,5-naphthalene-disulfonate,naphthalene-1-sulfonate, naphthalene-2-sulfonate, mandelate and thelike.

[0042] The term “base addition salt” refers to a salt of a compound offormula I prepared by reaction of a compound of formula I with a mineralor organic base. For exemplification of pharmaceutical base additionsalts see, e.g., Berge, S. M, Bighley, L. D., and Monkhouse, D. C., J.Pharm. Sci., 66:1, 1977. This invention also contemplates pharmaceuticalbase addition salts of compounds of formula I. The skilled artisan wouldappreciate that some compounds of formula I may be acidic in nature andaccordingly react with any of a number of inorganic and organic bases toform pharmaceutical base addition salts. Examples of pharmaceutical baseaddition salts are the ammonium, lithium, potassium, sodium, calcium,magnesium, methylamino, diethylamino, ethylene diamino, cyclohexylamino,and ethanolamino salts, and the like of a compound of formula I.

[0043] While all of the compounds of the present invention are useful,certain of the compounds are particularly interesting and are preferred.The following listing sets out several groups of preferred compounds. Itwill be understood that each of the listings may be combined with otherlistings to create additional groups of preferred embodiments.

[0044] a) R¹ is (CH₂)_(n)C(O)R²;

[0045] b) n is 0;

[0046] c) R² is NHR⁴, (optionally substituted C₁-C₄ alkyl)-optionallysubstituted phenyl or (CH₂)_(p)-optionally substituted heterocycle;

[0047] d) p is 3;

[0048] e) The compound is a pharmaceutical salt;

[0049] f) The compound is the hydrochloride salt;

[0050] g) The compounds of the Examples section;

[0051] h) The method where the mammal is a human;

[0052] i) The method where the oncolytic(s) is selected from:doxorubicin, daunorubicin, epirubicin, vincristine, and etoposide;

[0053] j) The method where the neoplasm is of the Wilm's type, bladder,bone, breast, lung(small-cell), testis, or thyroid or the neoplasm isassociated with acute lymphoblastic and myeloblastic leukemia,neuroblastoma, soft tissue sarcoma, Hodgkin's and non-Hodgkin'slymphomas, or bronchogenic carcinoma;

[0054] k) The formulation where the oncolytic(s) is selected from thegroup: doxorubicin, daunorubicin, epirubicin, vincristine, andetoposide.

[0055] The compounds of the present invention can be prepared by avariety of procedures, some of which are illustrated in the Schemesbelow. The particular order of steps required to produce the compoundsof formula I is dependent upon the particular compound beingsynthesized, the starting compound, and the relative lability of thesubstituted moieties.

[0056] Compounds of formula I may be prepared from compounds of formulaII as illustrated in Scheme 1 below where R¹ is as described supra.

[0057] Compounds of formula I may be prepared by dissolving orsuspending a compound of formula II in a suitable solvent, preferablydimethylformamide, and adding a suitable base, including potassiummethoxide, potassium tert-butoxide, potassium bis(trimethylsilyl)amide,potassium carbonate, sodium hexamethyldisilazane, and potassiumhexamethyldisilazane. The base is typically employed in a one to oneratio. However, as the skilled artisan would appreciate, a slight molarexcess, usually in about a 1.1 to about a 3 fold molar excess relativeto the compound of formula II, is acceptable.

[0058] The reactants are typically combined at a temperature from about0° C. to about 100° C. The reactants are preferably combined at roomtemperature, and the resulting solution is typically mixed for fromabout 5 minutes to about 18 hours, preferably from about 5 minutes toabout 1 hour.

[0059] Any protecting groups remaining in the cyclized compound offormula I may be removed as taught in Greene to provide additionalcompounds of formula L. Preferred choices of protecting groups andmethods for their removal may be found in the Preparations and Examplessections below.

[0060] Where R¹ is as described supra, compounds of formula II may beprepared according to Scheme 2.

[0061] Compounds of formula V may be converted to the corresponding acidhalide by methods well known to one skilled in the art. Compounds offormula II may be prepared by dissolving or suspending an acid halide ofa compound of formula V in a suitable solvent and adding a compound offormula IV in a suitable solvent. Triethylamine, N,N-diisopropylethylamine, dichloromethane, dimethylformamide, and mixtures thereof areconvenient solvents. This amide forming reaction is also preferably runin the presence of 4-dimethylaminopyridine (DMAP). The compound offormula V is preferably the corresponding carboxylic acid and isemployed in an equimolar amount, relative to the compound of formula IV,but a slight excess (about a 0.05 to about 0.15 molar excess) isacceptable. DMAP is employed in a catalytic fashion. For example, about5 molar percent to about 15 molar percent, relative to the compound offormula IV, is typically employed. A 10 molar percent is usuallypreferred.

[0062] Compounds of formula IV and V are well known in the art and tothe extent not commercially available, are readily synthesized bystandard procedures commonly employed in the art, see e.g. thePreparation section.

[0063] It should be recognized that the particular counterion forming apart of any salt of this invention is not of a critical nature, so longas the salt as a whole is pharmacologically acceptable and as long asthe counterion does not contribute undesired qualities to the salt as awhole.

[0064] The optimal time for performing the reactions of Schemes 1-2 canbe determined by monitoring the progress of the reaction viaconventional chromatographic techniques. Furthermore, it is preferred toconduct the reactions of the invention under an inert atmosphere, suchas, for example, argon, or, particularly, nitrogen. Choice of solvent isgenerally not critical so long as the solvent employed is inert to theongoing reaction and sufficiently solubilizes the reactants to effectthe desired reaction. The compounds are preferably isolated and purifiedbefore their use in subsequent reactions. Some compounds may crystallizeout of the reaction solution during their formation and then collectedby filtration, or the reaction solvent may be removed by extraction,evaporation, or decantation. The intermediates and final products offormula I may be further purified, if desired by common techniques suchas recrystallization or chromatography over solid supports such assilica gel or alumina.

[0065] The skilled artisan will appreciate that not all substituents arecompatible with all reaction conditions. These compounds may beprotected or modified at a convenient point in the synthesis by methodswell known in the art. For example, the R¹ substituent of the compoundsof formula IV may be a protecting group, which may be, removed duringthe synthesis of the compounds of formula I at any convenient point. Theprotecting group may be removed by methods well known in the art, seee.g. Greene, and R¹, R² and R³ may be added through standard chemicaltechniques, see e.g. Larock, Comprehensive Organic Transformations, pgs.785-820, 1640-1641, 1941-1949, and 1973-1976, VCH Publishers, New York,N.Y., 1999; and March J, Advanced Organic Chemistry, 1985, 3rd edition,page 377-378.

[0066] The following Preparations and Examples are provided to betterelucidate the practice of the present invention and should not beinterpreted in any way as to limit the scope of same. Those skilled inthe art will recognize that various modifications may be made while notdeparting from the spirit and scope of the invention. All publicationsmentioned in the specification are indicative of the level of thoseskilled in the art to which this invention pertains. The terms andabbreviations used in the instant Preparations and Examples have theirnormal meanings unless otherwise designated. For example “°C.”, “N”,“mmol”, “g”, “mL”, “M”, “HPLC”, “IR”, “MS(FD)”, “MS(IS)”, “MS(FIA)”,“MS(FAB)”, “MS(EI)”, “UV”, and “¹H NMR”, refer to degrees Celsius,normal or normality, millimole or millimoles, gram or grams, milliliteror milliliters, molar or molarity, high performance liquidchromatography, infra red spectrometry, field desorption massspectrometry, ion spray mass spectrometry, flow injection analysis massspectrometry, fast atom bombardment mass spectrometry, electron impactmass spectrometry, ultraviolet spectrometry, and proton nuclear magneticresonance spectrometry respectively. In addition, the absorption maximalisted for the IR spectra are only those of interest and not all of themaxima observed.

Preparations Preparation 1(6-{[3-(2-Chloro-6-fluorophenyl)-5-methyl-isoxazole-4carbonyl]-amino}-pyridin-2-yl)-aceticacid ethyl ester

[0067] Add Et₃N (0.7 ml, 5.06 mmol) to a solution of ethyl2-(2-Aminopyridin-6-yl)acetate (for preparation see Goto, Jiro; Sakane,Kazuo; Nakai, Yoshiharu; Teraji, Tsutomu; Kamiya, Takashi. J. Antibiot.(1984), 37(5), 532-45) (0.6 g, 3.33 mmol) and3-(2-chloro-6-fluoro-phenyl)-5-methyl-isoxazole-4-carbonyl chloride(0.694 g, 2.53 mmol) in dichloromethane (7 ml) and stir overnight.Dilute the reaction with dichloromethane, wash (H₂O then brine), dry(MgSO₄), filter, and concentrate. Column chromatography (silica gel,hexanesle EtOAc gradient) gives the title compound (0.81 g, 77%). MassSpectrum (FIA) (m/z) 418.2 [M+1]

EXAMPLES Example 1[6-(9-Chloro-3-methyl-4oxo-5H-isoxazolo[4,3-c]quinolin-5-yl)-pyridin-2-yl)-aceticacid ethyl ester

[0068] Add powdered K₂CO₃ (0.661 g, 4.8 mmol) to a solution of(6-{[3-(2-Chloro-6-fluorophenyl)-5-methyl-isoxazole-4-carbonyl]-amino}-pyridin-2-yl)-aceticacid ethyl ester (0.5 g, 1.2 mmol) in DMF (20 ml) under N₂ and stirredovernight. Dilute with EtOAc, wash (H₂O then brine), dry (MgSO₄),filter, and concentrate. Column chromatography (silica gel, hexanes/EtOAc gradient) gives the title compound (0.376 g, 79%). Mass Spectrum(FIA) (m/z) 398.1 [M+1]

Example 2[6-(9-Chloro-3-methyl-4-oxo-5H-isoxazolo[4,3-c]quinolin-5-yl)-pyridin-2-yl]-aceticacid

[0069] Heat[6-(9-Chloro-3-methyl-4-oxo-5H-isoxazolo[4,3-c]quinolin-5-yl)-pyridin-2-yl]-aceticacid ethyl ester (0.359 g, 0.9 mmol), MeOH (4.5 ml), THF (0.5ml), and 1NNaOH (1.8 ml, 1.8 mmol) at 50° C. for 2 h. Cool to room temperature,dilute with H₂O, and acidify (conc. HCl) to less than pH 3. Extract withEtOAc twice and wash the combined extracts (H₂O then brine), dry(MgSO₄), filter, and concentrate giving the title compound (0.338 g,100%). Use this material without further purification. Mass Spectrum(FIA) (m/z) 370.0 [M+1]

Example 32-[6-(9-Chloro-3-methyl-4-oxo-5H-isoxazolo[4,3-c]quinolin-5-yl)-pyridin-2-yl]-N-(3,4,5-trimethoxyphenyl)-acetamide

[0070] Add EDCI (0.016 g, 0.08 mmol), 3,4,5-trimethoxyaniline (0.014 g,0.075 mmol), and DMAP (0.001 g, 0.01 mmol) to a solution of[6-(9-Chloro-3-methyl-4-oxo-5H-isoxazolo[4,3]quinolin-5-yl)-pyridin-2-yl]-aceticacid (0.02 g, 0.05 mmol) in dichloromethane (1.25 ml) and DMF (0.5 ml).Stir overnight. Dilute with dichloromethane, wash (1.0 N HCl, H₂O,brine), dry (MgSO₄), filter, and concentrate. Column chromatography(silica gel, acetone/dichloromethane gradient) gives the title compound(0.024 g, 90%). Mass Spectrum (EIA) (m/z) 535.2 [M+1]

Example 42-[6-(9-Chloro-3-methyl-4-oxo-5H-isoxazolo[4,3-c]quinolin-5-yl)-pyridin-2-yl]-N-(2-methoxy-5-nitrophenyl)-acetamide

[0071][6-(9-Chloro-3-methyl-4-oxo-5H-isoxazolo[4,3-c]quinolin-5-yl)-pyridin-2-yl]-aceticacid (0.075 g, 0.2 mmol), EDCI (0.055 g, 0.3 mmol),2-Nitro-5-methoxyaniline (0.044 g, 0.26 mmol), DMAP (0.005 g, 0.04 mmol)in dichloromethane (4.5 ml) and DMF (0.75 ml) react for 5 h in a fashionsimilar to that of Example 3. Column chromatography (silica gel,acetone/dichloromethane gradient) gives the title compound (0.012 g,12%). Mass Spectrum (FIA) (m/z) 520.2 [M+1]

Example 52-[6-(9-Chloro-3-methyl-4-oxo-5H-isoxazolo[4,3-c]quinolin-5-yl)-pyridin-2-yl]-N-(3-methoxyphenyl)-acetamide

[0072][6-(9-Chloro-3-methyl4-oxo-5H-isoxazolo[4,3-c]quinolin-5-yl)-pyridin-2-yl]-aceticacid (0.075 g, 0.2 mmol), EDCI (0.055 g, 0.3 mmol), 3-methoxyaniline(0.03 ml, 0.26 mmol), DMAP (0.005 g, 0.04 mmol) in dichloromethane (4.5ml) and DMP (0.75 ml) react for 5.5 h in a fashion similar to that ofExample 3. Column chromatography (silica gel, acetone/dichloromethanegradient) gives the title compound (0.05 g, 53%).

[0073] Mass Spectrum (FIA) (m/z) 473.1 [M−1]

Example 62-[6-(9-Chloro-3-methyl-4-oxo-5H-isoxazolo[4,3-c]quinoln-5-yl)-pyridin-2-yl]-N-(3-nitrophenyl)-acetamide

[0074][6-(9-Chloro-3-methyl-4-oxo-5H-isoxazolo[4,3-c]quinolin-5-yl)-pyridin-2-yl]-aceticacid (0.075 g, 0.2 mmol), EDCI (0.055 g, 0.3 mmol), 3-nitroaniline(0.036 g, 0.26 mmol), DMAP (0.005 g, 0.04 mmol) in dichloromethane (4.5ml) and DNT (0.75 ml) react for 5.5 h in a fashion similar to that ofExample 3. Column chromatography (silica gel, acetonel dichloromethanegradient) gives the title compound (0.024 g, 24%). Mass Spectrum (WA)(m/z) 488.1 [M−1]

[0075] The compounds of the invention are inhibitors of MRP1. Thus, thecompounds of the invention may be used to inhibit any neoplasm havingintrinsic and/or acquired resistance, conferred in part or in total byMRP1, to an oncolytic or oncolytics. In other words, treatment of such aneoplasm with an effective amount of a compound of this invention willcause the neoplasm to be more sensitive to chemotherapy that wasrendered less efficacious by MRP1.

[0076] Vincristine, epirubicin, daunorubicin, doxorubicin, and etoposideare examples of oncolytics that are substrates of MRP1. See Cole, et.al., “Pharmacological Characterization of Multidrug ResistantMRP-transfected Human Tumor Cells”, Cancer Research, 54:5902-5910, 1994.Since MRP1 is ubiquitous in mammals, particularly humans, Nooter, K, et.al., “Expression of the Multidrug Resistance-Associated Protein (MRP)Gene in Human Cancers”, Clin. Can. Res., 1:1301-1310, (1995),chemotherapy whose goal is to inhibit a neoplasm employing any of thoseagents has the potential to be rendered less efficacious by MRP1. Thus,neoplasms of the bladder, bone, breast, lung(small-cell), testis, andthyroid and more specific types of cancer such as acute lymphoblasticand myeloblastic leukemia, Wilm's tumor, neuroblastoma, soft tissuesarcoma, Hodgkin's and non-Hodgkin's lymphomas, and bronchogeniccarcinoma may be inhibited with a combination of one or more of theabove oncolytics and a compound of this invention.

[0077] The biological activity of the compounds of the present inventionwas evaluated employing an initial screening assay, which rapidly andaccurately measured the activity of the tested compound in inhibitingMRP1 or MDR1. Assays useful for evaluating this reversing capability arewell known in the art. See, e.g., T. McGrath, et al., BiochemicalPharmacology, 38:3611, 1989; D. Marquardt and M. S. Center, CancerResearch, 52:3157, 1992; D. Marquardt, et al., Cancer Research, 50:1426,1990; and Cole, et. al., Cancer Research, 54: 5902-5910, 1994.

[0078] Assay for Reversal of MRP1-Mediated Doxorubicin Resistance andMDR1-Mediated Vincristine Resistance: HL60/ADR and HL60/VCR arecontinuous cell lines, which were selected for doxorubicin andvincristine resistance respectively by culturing HL60, a human acutemyeloblastic leukemia cell line, in increasing concentrations ofdoxorubicin or vincristine until a highly resistant variant wasattained.

[0079] HL60/ADR and HL60/VCR cells were grown in RPMI 1640 (Gibco)containing 10% fetal bovine serum (FBS) and 250 μg/ml GENTAMICIN™(Sigma). Cells were harvested; washed twice with assay medium (same asculture media); counted; and diluted to 2×10⁵ cells/ml in assay medium.Fifty microliters of cells were aliquoted into wells of a 96 well tissueculture plate. One column of each 96 well plate served as a negativecontrol and received assay medium containing no cells.

[0080] Test compounds and reference compounds were dissolved in dimethylsulfoxide (DMSO) at a concentration of 5 mM. Samples were diluted to 20μM in assay medium and 25 μl of each test compound was added to 6 wells.Assay standards were run in quadruplicate. Twenty-five microliters of0.4% DMSO was added to four wells as a solvent control. Assay media wasadded to all wells to achieve a final volume of 100 μl per well.

[0081] The plates were incubated at 37° C. for 72 hours in a humidifiedincubator with a 5% carbon dioxide atmosphere. Cell viability andvitality was measured by oxidation of a tetrazolium salt using standardconditions. The plates were incubated for 3 hours at 37° C. Absorbancewas determined at 490 nm using a microtitre plate reader.

[0082] The ability of a test compound to reverse the resistance ofHL60/ADR and HL60/VCR cells to doxorubicin was determined by comparisonof the absorbance of the wells containing a test compound in addition tothe oncolytic (doxorubicin) with the absorbance of wells containing theoncolytic without a test compound. Controls were used to eliminatebackground and to ensure the results were not artifactual. The resultsof the assay are expressed as percent inhibition of cell growth. Theoncolytic alone at the tested concentration does not usually inhibit thegrowth of HL60/ADR or HL60/VCR cells.

[0083] Representative compounds of formula I demonstrated a significanteffect in reversing the MRP1 multiple drug resistance. Many of thecompounds showed very significant enhancement of activity in combinationwith the oncolytic agent as opposed to the oncolytic agent alone. Inaddition, a large majority of the compounds tested displayed asignificant degree of selective inhibition of the HL60/ADR cell lineover the HL60/VCR cell line.

[0084] When administering an oncolytic in practicing the methods of thisinvention, the amount of oncolytic employed will be variable. It shouldbe understood that the amount of the oncolytic actually administeredwill be determined by a physician, in the light of the relevantcircumstances, including the condition to be treated, the chosen routeof administration, the actual oncolytic administered, the age, weight,and response of the individual patient (mammal), and the severity of thepatient's symptoms. Of course, the amount of oncolytic administeredshould be decided and closely monitored by that patient's physician.After deciding on the oncolytic or oncolytics to employ, “ThePhysician's Desk Reference®”, published by Medical Economics Company atMontvale, N.J. 07645-1742, is a helpful resource to the physician indeciding on amounts of the oncolytic to administer and is updatedannually.

[0085] Preferred formulations, and the methods of this inventionemploying those formulations, are those which do not contain anoncolytic. Thus, it is preferred to administer the compounds of thisinvention separately from the oncolytic. The oncolytics mentioned inthis specification are commercially available and may be purchased inpre-formulated forms suitable for the methods of this invention.

[0086] The compounds of formula I alone, or optionally in combinationwith an oncolytic, are usually administered in the form ofpharmaceutical formulations. These formulations can be administered by avariety of routes including oral, rectal, transdermal, subcutaneous,intravenous, intramuscular, and intranasal. Such formulations areprepared in a manner well known in the pharmaceutical art and compriseat least one active compound of formula I.

[0087] The present invention also includes methods employingpharmaceutical formulations, which contain, as the active ingredient,the compounds of formula I, and optionally an oncolytic, associated withpharmaceutical carriers. In making the formulations of the presentinvention the active ingredient(s) is usually mixed with an excipient,diluted by an excipient, or enclosed within such a carrier which can bein the. form of a capsule, sachet, paper or other container. When theexcipient serves as a diluent, it can be a solid, semi-solid, or liquidmaterial, which acts as a vehicle, carrier or medium for the activeingredient. Thus, the formulations can be in the form of tablets, pills,powders, lozenges, sachets, cachets, elixirs, suspensions, emulsions,solutions, syrups, aerosols (as a solid or in a liquid medium),ointments containing for example up to 10% by weight of the activecompound, soft and hard gelatin capsules, suppositories, sterileinjectable solutions, and sterile packaged powders.

[0088] In preparing a formulation, it may be necessary to mill theactive compound(s) to provide the appropriate particle size prior tocombining with the other ingredients. If the active compound(s) issubstantially insoluble, it ordinarily is milled to a particle size ofless than 200 mesh. If the active compound(s) is substantially watersoluble, the particle size is normally adjusted by milling to provide asubstantially uniform distribution in the formulation, e.g., about 40mesh.

[0089] Some examples of suitable excipients include lactose, dextrose,sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate,alginates, tragacanth, gelatin, calcium silicate, microcrystallinecellulose, polyvinylpyrrolidone, cellulose, water, syrup, and methylcellulose. The formulations can additionally include: lubricating agentssuch as talc, magnesium stearate, and mineral oil; wetting agents;emulsifying and suspending agents; preserving agents such as methyl- andpropylhydroxybenzoates; sweetening agents; and flavoring agents. Theformulations of the invention can be formulated so as to provide quick,sustained or delayed release of the active ingredient afteradministration to the patient by employing procedures known in the art.

[0090] The formulations are preferably formulated in a unit dosage form,each dosage containing from about 5 to about 100 mg, more usually about10 to about 30 mg, of each active ingredient. The term “unit dosageform” refers to physically discrete units suitable as unitary dosagesfor human subjects and other mammals, each unit containing apredetermined quantity of active material calculated to produce thedesired therapeutic effect, in association with a suitablepharmaceutical excipient.

[0091] The compounds of formula I are effective over a wide dosagerange. For example, dosages per day normally fall within the range ofabout 0.5 to about 30 mg/kg of body weight. In the treatment of adulthumans, the range of about 1 to about 15 mg/kg/day, in single or divideddose, is especially preferred. However, it will be understood that theamount of the compound actually administered will be determined by aphysician, in the light of the relevant circumstances, including thecondition to be treated, the chosen route of administration, the actualcompound administered, the age, weight, and response of the individualpatient, and the severity of the patient's symptoms, and therefore theabove dosage ranges are not intended to limit the scope of the inventionin any way. In some instances dosage levels below the lower limit of theaforesaid range may be more than adequate, while in other cases stilllarger doses may be employed without causing any harmful side effect,provided that such larger doses are first divided into several smallerdoses for administration throughout the day.

[0092] For preparing solid formulations such as tablets, the principalactive ingredient(s) is mixed with a pharmaceutical excipient to form asolid preformulation composition containing a homogeneous mixture of acompound of the present invention. When referring to thesepreformulation compositions as homogeneous, it is meant that the activeingredient(s) is dispersed evenly throughout the formulation so that theformulation may be readily subdivided into equally effective unit dosageforms such as tablets, pills and capsules. This solid preformulation isthen subdivided into unit dosage forms of the type described abovecontaining from 0.1 to about 500 mg of the active ingredient of thepresent invention.

[0093] The tablets or pills of the present invention may be coated orotherwise compounded to provide a dosage form affording the advantage ofprolonged action. For example, the tablet or pill can comprise an innerdosage and an outer dosage component, the latter being in the form of anenvelope over the former. The two components can be separated by entericlayer, which serves to resist disintegration in the stomach and permitthe inner component to pass intact into the duodenum or to be delayed inrelease. A variety of materials can be used for such enteric layers orcoatings, such materials including a number of polymeric acids andmixtures of polymeric acids with such materials as shellac, cetylalcohol, and cellulose acetate.

[0094] The novel formulations which are liquid forms may be incorporatedfor administration orally or by injection and include aqueous solutions,suitably flavored syrups, aqueous or oil suspensions, and flavoredemulsions with edible oils such as cottonseed oil, sesame oil, coconutoil, or peanut oil, as well as elixirs and similar pharmaceuticalvehicles.

[0095] Formulations for inhalation or insufflation include solutions andsuspensions in pharmaceutical, aqueous or organic solvents, or mixturesthereof, and powders. The liquid or solid formulations may containsuitable pharmaceutical excipients as described supra. Preferably theformulations are administered by the oral or nasal respiratory route forlocal or systemic effect. Compositions in preferably pharmaceuticalsolvents may be nebulized by use of inert gases. Nebulized solutions maybe breathed directly from the nebulizing device or the nebulizing devicemay be attached to a face mask, tent, or intermittent positive pressurebreathing machine. Solution, suspension, or powder formulations may beadministered, preferably orally or nasally, from devices, which deliverthe formulation in an appropriate manner.

[0096] The following formulation examples are illustrative only and arenot intended to limit the scope of the invention in any way. “Activeingredient(s)” means a compound according to formula I or apharmaceutical salt thereof optionally with one or more oncolytics.

[0097] Another preferred formulation employed in the methods of thepresent invention employs transdermal delivery devices (“patches”). Suchtransdermal patches may be used to provide continuous or discontinuousinfusion of the compounds of the present invention in controlledamounts. The construction and use of transdermal patches for thedelivery of pharmaceutical agents is well known in the art. See, e.g.,U.S. Pat. No. 5,023,252, issued Jun. 11, 1991, herein incorporated byreference. Such patches may be constructed for continuous, pulsatile, oron demand delivery of pharmaceutical agents.

[0098] Frequently, it will be desirable or necessary to introduce thepharmaceutical formulation to the brain, either directly or indirectly.Direct techniques usually involve placement of a drug delivery catheterinto the host's ventricular system to bypass the blood-brain barrier.One such implantable delivery system, used for the transport ofbiological factors to specific anatomical regions of the body, isdescribed in U.S. Pat. No. 5,011,472, issued Apr. 30, 1991, which isherein incorporated by reference.

[0099] Indirect techniques, which are generally preferred, usuallyinvolve formulating the compositions to provide for drug latentiation bythe conversion of hydrophilic drugs into lipid-soluble drugs orprodrugs. Latentiation is generally achieved through blocking of thehydroxy, carbonyl, sulfate, and primary amine groups present on the drugto render the drug more lipid soluble and amenable to transportationacross the blood-brain barrier. Alternatively, the delivery ofhydrophilic drugs may be enhanced by intra-arterial infusion ofhypertonic solutions, which can transiently open the blood-brainbarrier.

1. A compound of formula I:

wherein: R¹ is hydrogen, C₁-C₄ alkyl optionally substituted 1 time witha hydroxy group, (CH₂)_(n)C(O)R², (C₁-C₄ alkyl)NH₂, (CH₂)_(n)NHC(O)R³,C₁-C₄ alkyl optionally substituted 1 time with a hydroxy group)- phenyloptionally substituted 1 or 3 times independently with a C₁-C₆ alkyl,C₁-C₄ alkoxy, halo, benzyl, phenyl, trifluoromethoxy, nitro, or an oxogroup, or heterocycle optionally substituted 1 or 2 times independentlywith a C₁-C₆ alkyl. C₁-C₄ alkoxy, halo, benzyl, phenyl, trifluoromethyl,or an oxo group; n is 0, 1, or 2; p is 0, 1, 2, 3, or 4; R² is C₁-C₄alkoxy, hydroxy, C₁-C₄ alkyl optionally substituted 1 time with ahydroxy group)- phenyl optionally substituted 1 or 3 times independentlywith a C₁-C₆ alkyl, C₁-C₄ alkoxy, halo, benzyl, phenyl,trifluoromethoxy, nitro or an oxo group, (CH₂)_(p)— heterocycleoptionally substituted 1 or 2 times independently with a C₁-C₆ alkyl,C₁-C₄ alkoxy, halo, benzyl phenyl, trifluoromethyl, or an oxo group,NHR⁴, or (CH₂)_(p)—O— heterocycle optionally substituted 1 or 2 timesindependently with a C₁-C₆ alkyl C₁-C₄ alkoxy halo benzyl, phenyl,trifluoromethyl, or an oxo group; R³ is C₁-C₄ alkoxy, phenyl optionallysubstituted 1 or 3 times independently with a C₁-C₆ alkoxy, halo,benzyl, phenyl, trifluoromethoxy, nitro, or an oxo group, C₁-C₄ alkyloptionally substituted 1 time with a hydroxy group)- phenyl optionallysubstituted 1 or 3 times independently with a C₁-C₆ alkyl, C₁-C₄ alkoxyhalo, benzyl, phenyl, trifluoromethoxy, nitro, or an oxo group, or(CH₂)_(p)— optionally substituted heterocycle optionally substituted 1or 2 times independently with a C₁-C₆ alkyl, C₁-C₄ alkoxy, halo, benzyl,phenyl, trifluoromethyl, or an oxo group; R⁴ is (CH₂)_(p)— optionallysubstituted phenyl optionally substituted 1 or 3 times independentlywith a C₁-C₆ alkyl, C₁-C₄ alkoxy, halo, benzyl, phenyl, trifluoromethoxynitro, or an oxo group, or (CH₂)_(p)— optionally substituted heterocycleoptionally substituted 1 or 2 times independently with a C₁-C₆ alkyl,C₁-C₄ alkoxy halo, benzyl, phenyl, trifluoromethyl, or an oxo group; ora pharmaceutical salt thereof. 2-3. (canceled)
 4. A method of inhibitingMRP1 which comprises administering to a mammal in need thereof aneffective amount of a compound of claim
 1. 5-7. (canceled)
 8. A methodof inhibiting a resistant neoplasm, or a neoplasm susceptible toresistance, in a mammal which comprises administering to a mammal inneed thereof an effective amount of a compound of claim 1 in combinationwith an effective amount of one or more oncolytic agents. 9-12.(canceled)
 13. A pharmaceutical formulation comprising a compound ofclaim 1 or a pharmaceutical salt thereof in combination with one or morepharmaceutical carriers, diluents, or excipients therefor. 14.(canceled)
 15. (canceled)